The cardiovascular system is among the earliest organs to form in mammals. De novo gene expression of smooth muscle contractile protein genes appears in the heart-forming regions providing molecular markers for promyocardial cells Well before overt cardiac morphogenesis (Colas et al., 2000). Robust expression of the smooth muscle α-actin (SMA) gene marked the onset of differentiation of cardiac cells and represented the first demonstration of coexpression of both smooth muscle and striated α-actin genes within myogenic cells (Ruzicka and Schwartz, 1988). Cardiovascular smooth muscle differentiation depends upon the integration of complex signaling inputs with activation of serum response factor (SRF)-dependent transcription. SRF contains a highly conserved DNA-binding/dimerization domain termed the MADS box that integrates intracellular signals and assists as a docking surface for the binding of cofactors, which may confer the regulation of specific gene programs (reviewed in Reecy et al., 1999; Gineitis and Treisman, 2001). The knockout of the murine SRF gene locus supports the observation that SRF is absolutely required for the appearance of muscle mesoderm during mouse embryogenesis (Arsenian et al., 1998). While numerous studies have established the importance of an evolutionarily-conserved, SRF-binding cis element termed an SRE or CArG box [CC(AT)6GG; SEQ ID NO:1] in control of smooth muscle cell (SMC)-specific gene expression (Li et al., 1997; Kim et al, 1997; Belaguli et al., 1999; Mack and Owens, 1999; Miano et al., 2000), it is clear that SRF-SRE interactions, by themselves, are not sufficient to direct SMC differentiation from committed progenitor cells.
Proteins that act to organize higher-order multi-protein complexes that contain SRF and stimulate SMC-restricted gene transcription in an SRE-dependent and developmental signal-responsive manner have been sought. Recently, Sepulveda et al. (2002) demonstrated that the expression of SRF, Nkx2-5 and GATA4 was central for activation of embryonic cardiac α-actin gene activity. Nishida et al. (2002) showed the combinatorial interaction of SRF, Nkx-3.2 and GATA-6 activated promoters of smooth muscle genes. Wang et al. (2001) showed that myocardin, a SAP factor, enriched during cardiogenesis, also served as a powerful SRF co-accessory factor (Wang et al., 2001). However, the precise combination of regulatory factors that fosters distinct cardiac and smooth muscle cell lineages has not been previously elucidated.
Of interest in this regard are the LIM domain-containing members of the cysteine-rich protein (CRP) family. LIM domains are double zinc finger-like structures that mediate protein-protein interactions and may target proteins to distinct subcellular locations and mediate assembly of multimeric protein complexes (Dawid et al., 1995; Louis et al., 1997). Each member of the CRP family contains two LIM domains with associated glycine-rich repeats. Three members of the CRP family (CRP1, CRP2/SmLIM and CRP3/MLP) have been characterized in vertebrates (Liebhaber et al., 1990; Weiskirchen et al., 1993; Arber et al., 1994) and all three appear to have comparable functions in different cell types. CRP1 is expressed in multiple adult organs, including those enriched in smooth muscle such as arteries, stomach, gizzard and intestine (Henderson, et al, 1999). CRP2 is expressed mainly in vascular SMCs and is down-regulated upon their dedifferentiation and proliferation in response to stress or injury, such as occurs in arteriosclerosis (Jain et al., 1996). Expression of CRP3 is limited to striated muscle cells of heart and skeletal muscle, and CRP3-deficient mice develop heart failure soon after birth (Arber et al., 1997).
One distinguishing feature of the three CRPs is their association with adhesion plaques and with filamentous actin. These proteins do not bind actin directly, but all three interact with both the actin cross-linking protein α-actinin and the adhesion plaque protein zyxin and may regulate the stability and structure of adhesion complexes. (Schmeichel and Beckerle, 1994; Arber and Caroni, 1996; Pomies et al., 1997) However, in a specific embodiment, this cytoplasmic role for the CRP proteins is not their only, or possibly even their most critical, function during cell differentiation. The present invention is directed to methods and compositions related to the CRP proteins for cell differentiation and, in some embodiments, for cardiac disease therapy.
Serum response factor expression is required for induction of a smooth muscle phenotype in mesenchymal cells and is sufficient for transforming growth factor-beta-mediated smooth muscle induction (Hirschi et al., 2002).